Chromosomal translocations bring two previously unlinked regions of the genome together and in certain cases can result in disease by inducing synthesis of a novel fusion protein. This phenomenon is significant when the breakpoint of the translocation affects an oncogene, for example, and results in cancer.
The exemplary BCR/ABL fusion protein is a constitutively-active tyrosine kinase present in patients with chronic myeloid leukemia (CML) as well as some patients with acute lymphoblastic leukemia (ALL) with the Philadelphia chromosome (9:22) translocation. Recently, this fusion tyrosine kinase protein has been successfully targeted for therapy through the use of specific tyrosine kinase inhibitors such as STI571 (Gleevec). Despite the success of such a drug, a significant number of patients (10-30%) have demonstrated poor response, as well as development of resistance to therapy. Therefore, it is increasingly important to be able to monitor the activity of the BCR/ABL protein in response to therapy as a means to monitor efficacy and response, as well as a potential diagnostic tool, or as well as predicting response or prognosis. Discovery of resistance to therapy by Gleevec due to mutational abnormalities within the BCR/ABL gene, or other exemplary mechanisms, has led to the emergence of new drugs such as BMS-354825 and AMN107 to target these resistant forms of the BCR/ABL protein. Therefore, there is significant need to develop means of monitoring effects of Gleevec therapy at early stages. Direct evaluation of BCR/ABL protein activity (phosphorylation) or levels has been limited due to the large size of the protein (190 or 210 kD). Evaluation usually has been largely based on western blot or immunoprecipitation techniques, both of which are difficult to implement in clinical laboratories for patient samples. As a result, downstream modulators such as AKT and CRK-L proteins have been used to monitor BCR/ABL activity. Although monitoring of such downstream signaling molecules has proved effective, mainly through western blot analysis, a direct and quantitative measure of the levels and activation of the BCR/ABL protein itself is desired.
The LightCycler quantification kits (Roche Molecular Biochemicals) reverse transcribe cDNA from RNA and a partial fragment of the mRNA is amplified using specific primers. Following this, the amplicon is detected using specific hybridization probes.
Talpaz et al. (2000) describe autoantibodies to Abl and Bcr proteins in Philadelphia (Ph) chromosome-positive leukemias.
Valk et al. (2003) describe qualitative RT-PCR assays and real-time quantitative RT-PCR assays using commercially available sequence detection systems, particularly for detecting increased sensitivity minimal residual disease studies.
van Denderen et al. (1990) regards polyclonal antiserum raised against a synthetic peptide corresponding to the bcr-abl junction in P190bcr-abl, and immunoprecipitation utilizing antibodies directed thereto identified specificity against P190bcr-abl but not P210bcr-abl.
Wallace et al. (2003) describe two-stage multiplexing comprising polymerase chain reaction with multiplex detection on spectrally addressable liquid bead microarrays, such as for identifying seven exemplary fusion transcripts in lymphoblastic leukemia.
U.S. Pat. No. 5,369,008 relates to detecting BCR-ABL in a sample by assaying for binding of an antibody to BCR-ABL, particularly upon binding of the antibody to the SH2 region of the ABL gene product.
WO 95/15331 concerns detection of specific fusion proteins, such as by contacting a sample with two antibodies, each of which are capable of binding to a different region of the fusion protein.
U.S. Pat. No. 6,686,165 regards detecting chromosomal aberrations by binding separately-targeted probes for a tumor-specific protein and detecting them by flow cytometry. In specific embodiments, the chromosomal aberrations are employed as targets for monitoring the level of residual disease during and after therapy.
A specific, and in certain aspects, quantitative measure of the levels and activation of a fusion protein itself is desired and provided by the novel disclosure of the present invention. The present invention also employs novel methods for monitoring therapy for a disease.